Practical considerations for quantitative and reproducible measurements with stimulated Raman scattering microscopy

Stimulated Raman scattering (SRS) microscopy provides rapid label-free 3D chemical imaging with wide-ranging applications including histology, pharmacokinetic studies, and materials characterisation. SRS microscopy has seen a steady increase in utilisation since the early 2000s and has become more accessible due to the increase in availability of facilities, and the development of user-friendly instrumentation. Although some complete SRS systems are now commercially available, many instruments are home-built with highly varied laser sources, optics, and detection mechanisms. Signal intensity is also dependent on the effective spatiotemporal overlap of two (or more) laser beams, thus any drift in alignment can result in variable performance. Currently there is a lack of standard procedures or reference materials for SRS, which has important implications on reproducibility and consistency. These concerns are particularly relevant to the comparison of data from the same instrument at different times and instrument settings as well as between different instruments and laboratories. This tutorial-style review presents the most important practical considerations for sample preparation, instrument set-up, image acquisition and data analysis to obtain reproducible SRS measurements.